For some 30 years, desired heterologous proteins have been produced in microorganisms. However, having introduced the necessary coding sequence and obtained expression, much still remains to be done in order to optimise the process for commercial production. One area of interest concerns strain improvement, that is to say finding or making strains of the host microorganism which enable the protein to be made in higher yields or better purity, for example.
To increase the yield, once a good expression system has been devised, one might envisage trying to increase the copy number of the coding sequence, or to increase the quantity or stability of the mRNA, or to improve folding and/or secretion of the protein or to decrease the degradation of the protein. However, the desired effect of increased expression will only be seen if the limiting factor(s) is targeted.
Therefore, what is required is a host strain which allows the yield of a desired protein, such as a heterologous protein, to be increased. The inventors have surprisingly identified that mutation of NOT4 (also known as MOT2) results in such an increased yield.
Not4 is a ubiquitin-ligating enzyme and is part of the Ccr4-Not complex. The Ccr4-Not complex is conserved in eukaryotic cells, and in yeast the complex consists of 9 core subunits: Ccr4, Caf1, Caf40, Caf130, Not1, Not2, Not3, Not4 and Not5 (Collart, 2003, Global control of gene expression in yeast by the Ccr4—Not complex. Gene 313: 1-16; Bai et al., 1999, The CCR4 and Caf1 proteins of the Ccr4—Not complex are physically and functionally separated from Not2, Not4, and Not5. Mol. Cell. Biol. 19: 6642-6651). The complex has been proposed to function as a central switchboard that can interpret signals from the environment and coordinate all levels of gene expression to economically respond to the signal (Collart, 2012, The Ccr4-Not complex. Gene 492(1): 42-53). It is thought that Not proteins (Not1, Not2, Not3, Not4) are necessary for assembly of the RNA polymerase II complex, which suggests a global role in transcription regulation (Collart, 1994, Not1(cdc39), Not2(cdc36), Not3, and Not4 encode a global-negative regulator of transcription that differentially affects tata-element utilization. Genes & Development 8(5): 525-537; Collart, 2012, as cited above).
Recently a co-crystal structure suggested how the C-terminal region of Not4 wraps around a HEAT-repeat region of Not1, the scaffold protein in the Ccr4-Not complex (Bhaskar, 2015, Architecture of the ubiquitylation module of the yeast Ccr4-Not complex. Structure 23(5): 921-8).